The Cloning, Analysis Of The Full-length CDNA Of EPSPs From Brassica Campestris And Its Expression In Escherichia Coli

In bacteria, fungi, algae, apicomplexan parasites and plants, there is the shikimate pathway, of which the 5-enolpyruvylshikimate-3-phosphate svnthase (EPSPs) catalyses the conversion of phosphoenolpyruvate (PEP) and shikimate-3- phosaphate (S3P) into5-enolpyruvylshikimate-3-phosphate (EPSP) during the sixth step. Therefore, EPSPs is very significant in bacteria, fungi, algae, and apicomplexan parasitesand plants.According to the unpublished data of our lab and the consensus of EPSPs from Brassica napus and Orychophragmus violaceus, two primers, 5"-ATGGCGCAAGCTAGCAGAATC-3' and 5'-GCATTGACACGAAC AGGAGGAC-3', were designed. The 630bp 5'-cDNA was cloned from Brassica campestris and then connected to its 3'-cDNA. The full-length cDNA of EPSPs has 1726bp and its 1545bp ORF codes 514 amino acids. Blasted with other 8 plant EPSPs cDNA, the identity of EPSPsfrom B. campestris to those of B. napus, O. violaceus, Arabidopsis thaliana, Lycopersicon esculentum, Nicoiiana tabacum, Pecunia hybrida, Diciliptera chinensis, and Oryza sativa respectively is 93%, 92%, 88%, 68%. 68%, 67&, 65%, 64%. The homologous comparison of the putative amino acid sequence with the EPSPs described as above, shows that the identity of EPSPs from B. campestris to those of B. napus, O. violaceus,A. thaliana, L. esculentum, N. tabacum, P. hybrida, D. chinensis, and O. sativa, respectively is 95%, 95%, 92%, 74%, 73%, 72%, 72%, 71%. As far as the cDNA sequence of EPSPs is concerned, B. campestris is most relative to B.napus, then O.violaceus, and least relative to O. sativa, which is coherent to the traditional morphologic classification conclusion. The ORF fragment was amplified from the B. campestris by PT-PCR, using two primers, 5'-CGGAATTCATGGCGCAAGCTAGC AGAATC-3' and 5'-TTAGTGCTTTGTGATACTTTCAAGGAC-3'and then inserted into the expression vector, GTK, which is derived from the pGEX-2T. After induced by IPTG in E.Coli ,DH5 a , the fusion protein GST-EPSPs was expressed effectively. It is showed that, as to the expression efficiency, 0.1mM IPTG is better than 0.3mM, 0.5mM,lmM IPTG, 37℃ is better than 30℃, and 1-2 hour induction time is the best. And it has also been found that the expression of the fusion protein is lower than that of GST and it is to be elucidated later.